1. Field of the Invention
The present invention relates to clinical analyses of aqueous samples in which a reducible substrate is used as an indicator to detect the presence of bacteria or leukocytes. In particular, it relates to a method of improving the specificity of reduction assays by removing the leukocyte response through selective lysis of leukocyte cells.
2. General History
The increased availability of specific and effective antibiotics has produced a commensurate need for more rapid methods of clinical diagnosis of infection. The standard technique of diagnosing infection was to culture a sample suspected of containing the infecting bacteria, but this is a time-consuming process requiring skilled personnel. This has lead to a search for chemical methods of detecting and quantifying bacteria in body fluids.
One known method involves the use of a reducible chromogen as an indicator. This method known as Uroscreen, uses a reagent, triphenyltetrazolium cloride, and is described in "A Simple Semiquantitative Diagnostic Screening Method for Presumptive Bacteriuria," Pfizer, Diagnostics Division, New York (Brochure), 1974. Uroscreen.TM. is a dry, buffered reagent which is reduced in the presence of large numbers of bacteria to a pink-red insoluble precipitate after four hours of incubation. The manufacturer notes that highly colored or bloody urine may obscure the results.
There is a serious problem with this type of procedure. Clinical infections are often accompanied by a significant number of leukocytes which would additionally be present in samples for analysis. Furthermore, a significant number of leukocytes may be present in samples such as urine even in the absence of infection. Unfortunately, leukocytes, are also capable of reducing known chromogens such as those found in the Uroscreen.TM. method and thus, leukocyte reduction of the chromogen produces false positive results. This renders the known bacterial assays unreliable and makes quantification impossible.
Thus, the problem is to provide a fast and leukocyte interference-free assay of bacteria.